作者: Sophie Coudeyras, Gwendoline Jugie, [...], and
Christiane Forestier
日期:103.5.8
The ability of a probiotic Lactobacillus
rhamnosus strain (Lcr35) to adhere to cervical
and vaginal cells and to affect the viability of
two main vaginosis-associated pathogens,
Prevotella bivia, Gardnerella vaginalis, as well
as Candida albicans was investigated
• Bacterial vaginosis (BV) is the most frequent
vaginal infectious disorder in women of
childbearing age with prevalences ranging from
10% to 50%
• The cause of BV remains poorly understood,
and no specific infectious agents have been
identified. However, the disorder is
characterized by modifications of the genital
tract microflora, including a reduction in or
absence of lactobacillus colonization and
overgrowth of several anaerobic bacteria
• Different mechanisms are potentially involved
in the activity of Lactobacilli against
pathogens, including the competitive
exclusion of genitourinary pathogens from
receptors present on the surface of the
epithelial cells. Under healthy conditions,
cervicovaginal cells are constantly exposed to
the normal vaginal microbiota.
• The recommended treatment regimens for
vaginal infections are oral or intravaginal
antibiotics but these conventional treatments
are associated with frequent recurrences.
• Lactobacilli can restore ecological balance in
the vagina by controlling the infectivity of
pathogenic microbes
• L. acidophilus and L. rhamnosus species these
probiotics represent an effective and safe
method for treating women with BV can be
drawn.
• The behavior of the probiotics in the vaginal
tract is likely to be strain specific and therefore,
it is important to determine the characteristics
of the strain to be used as a therapeutic agent
• The purpose of this study was to determine
the in vitro adherence of a well characterized
L. rhamnosus probiotic strain,Lcr35,and its
ability to inhibit growth of three
vaginosisassociated pathogens.
• We used immortalized morphologically and
functionally distinct epithelial cell lines from
normal endocervix, ectocervix, and vagina to
characterize Lcr35 epithelial interactions
pertinent to the lower female genital tract and
determined its antimicrobial activity against
Prevotella bivia, Gardnerella vaginalis, and
Candida albicans in coculture experiments.
region
1. vagina (VK2/E6E7 ATCC-CRL-2616),ectocervix
2. (Ect1/E6E7 ATCC-CRL-2614),
3. endocervix(End1/E6E7 ATCC-CRL-2615)
1.keratinocyte serumfree
2.medium (Gifco BRL 17005-042)
3.human recombinant EGF (0.1 ng/mL),
bovine pituitary
4.extract (0.05 mg/mL)
5.calcium chloride (0.4mM)
1.Adhesion of the Lcr35 was assayed by seeding
cell lines in 24-well tissue culture plates at 2.5
× 105 epithelial cells/well and allowing them
to grow to complete confluence(105
cells/well).
2. gentle washing of the cell monolayer, the
adhesion capacity of Lcr35 was determined by
adding 105multiplicity of infection (MOI,1),
106 (MOI, 10), and107 (MOI,
100)bacteriafrom an overnight culture in
deMan, Rogosa
3.Sharpe (MRS) agar medium. Bacterial cells
were previously washed in phosphate buffered
saline and resuspended in the cell culture
medium
4. Adhesion was monitored after 1 and 3
hours of incubation carried out at 37◦C under
5% CO2
5.The monolayers were washed three times with
1 mL of Dulbecco's phosphate buffered saline,
detached by addition of 0.1% TritonX-100
solution and the number of viable bacteria
determined by plating serial dilutions of the
suspensions onto MRS agar plates.
6. qualitative analysis, the cell monolayers and
the bacteria were methanol fixed and stained
by addition of a 10% Giemsa solution.
1. Candida albicans ATCC10231
2. Prevotella bivia ATCC29303
3. Gardnerella vaginalis ATCC14018
1. Sabouraud broth(Candida) or brain
2. brain heart infusion supplemented with yeast
extract (1%)
3. maltose (0.1%)
4. glucose (0.1%)
5. horse serum (10%)
Adherence of L.
rhamnosus Lcr35 to
vaginal, ecto-and
endocervical cells.
Epithelial cells
were incubated
with three
different bacterial
inocula and
incubated for 1
hour
Microscopical observations of Giemsa-stained
preparation showed typical chains of Lcr35 randomly
dispersed on the cell surface
Giemsa-stains from
adherence assays
performed for the
Experiment
(a)Vaginal
(b)Ectocervical cells
The antagonist effect of Lcr35 against three
main pathogens, P. bivia, G. vaginalis, and C.
albicans,was assessed incoculture assays and
compared with the growth ability of each
pathogen in the same culture medium(Figure 3).
Effect of Lcr35 on the viability of
Prevotella bivia (a), Gardnerella
vaginalis (b), and Candida albicans
(c) as a function of the time of
coculture
Lcr35 at 37◦C for 24 hours and the
colony forming unit mL−1 was
determined after 4, 8, and 24 hours
of incubation by plating onto
appropriate media
When the viable bacteria in the mixed suspension
were counted over a longer period of time,
bactericidal activity was detected between 8 and
24 hours of incubation for all pathogens with the
Prevotella strain being the most susceptible (4log10 units decrease in the number of viable cells).
1.Lactobacillus species in the female urogenital system act
as a barrier to infection and contribute to the control of
the vaginalmicrobiota by competing with
othermicroorganisms for adherence to epithelial cells,
displacing pathogen biofilm, and/or inhibiting the
growth of potential pathogens
2. We can thus speculate that adhesion assays performed
with this material reproduce more faithfully the in vivo
situation than experiments performed with any cell line
derived from human carcinoma of the lower genital tract
mucosa. Using these cell lines, we observed specific
adhesion of an L. Adhesion occurred even at a low MOI
(1:1) and within less than 1 hour of contact, which
corresponds to a highly dynamic process.
3. strains of Lactobacilli were able to disrupt G. vaginalis
preformed biofilms .It would therefore be interesting
to test the biofilm activity of Lcr35 against G. vaginalis
and to determine if the sessile form of Lcr35 also exhibits
antibacterial activity against G. vaginalis in mixed biofilm
assays.
4. The antagonist activity of Lcr35 was not limited to
bacterial pathogens since the strain was also able to reduce
the viability of C. albicans, which is the species most often
associated with candidiasis,By interfering with Candida
overgrowth in the patients' intestinal or vaginal tract,
Lactobacilli could provide colonization resistance and
maintain low numbers of yeasts, especially when
administered together with antibiotics.
5. Petricevic and Witt recently showed in a clinical
study that topical administration of Lcr35 enhances
the restoration of the vaginal flora after antibiotic
treatment of BV. Thus, it might be an excellent
candidate for use as a prophylactic agent, taken orally
or applied topically.
Lactobacilli may be of prophylactic value in
preventing or curing genitourinary system
infections in women, rhamnosus Lcr35 would
be a good candidate as a protective agent
against both bacterial vaginosis and Candida
vaginitis since it was able to adhere to
vaginal and cervical cells and to antagonist
the growth of vaginosis-associated
pathogens. Clinical studies are now required
to assess the in vivo efficacy of such a
therapy